Single nucleotide polymorphisms (SNPs) are believed to be of significant potential importance for personalised medicine; it is possible that certain SNPs which result in single amino acid changes may give rise to variation in patient responsiveness to certain treatment regimes. SNPs may also be of importance in the development of cancers and other cell proliferative disorders, again with mutations in somatic cells activating certain oncogenes.
It is important to be able to determine which SNPs are present in a patient. The melting temperature (Tm) of double stranded DNA depends on the extent of base pair hybridisation; where there is a mismatch between a probe and a target sequence (for example, due to the presence of a SNP) then the Tm will differ compared with when there is no mismatch. Methods involving detecting changes in melting temperature (Tm) of DNA hybridisation between a specific oligonucleotide probe and a gene target when a SNP is present are known. However, somatic cell mutations suffer from the problem that the mutant sequence is likely to be present only in a very few copies in a particular sample, with the wild type sequence predominating. It would be useful to be able to selectively amplify the mutant sequence, for example by polymerase chain reaction (PCR) type techniques.
U.S. Pat. No. 5,849,497 describes specific inhibition of PCR amplification using an oligonucleotide blocking probe which hybridises to a region of interest, but which also includes 3′ mismatched bases, which do not hybridise. This mismatch prevents strand extension during PCR. This method is said to be useful for preventing amplification of a specific known sequence. The patent suggests use of the method of prevent amplification of an allele including an inserted sequence by designing a blocking probe to the inserted region. However, this patent does not teach methods for selectively amplifying one SNP variation compared with another. Further, it is necessary to know the target sequence of interest.
Bender et al, Biotechniques 2007, vol 42 no 5, pp 609-614, describe the use of a PNA (peptide nucleic acid) blocking probe to prevent DNA-mediated PCR product formation in prokaryotic RT-PCR. The probe is designed to bind to a region in the genomic DNA which is not present in cDNA generated from mRNA; thus, only cDNA will be amplified, not genomic DNA.
Vestheim and Jarman, Frontiers in Zoology 2008, 5:12, describe the use of blocking primers to prevent PCR amplification of krill DNA sequences and allow amplification of prey DNA from krill stomach content samples. The primers are designed against krill rDNA sequences.
It is among the objects of the present invention to provide a method whereby a variant SNP sequence can be selectively amplified from a sample containing both the variant and the wild type sequence. It is further among objects of aspects of the invention to provide a method whereby amplified sequences can be analysed for SNP content.